Antigenic peptides

ABSTRACT

An improved antigenic peptide where the amino acid sequence is modified by replacing one or more amino acids with glycine.

This is a Continuation of application Ser. No. 08/763,302, filed Dec.10, 1996, now U.S. Pat. No. 5,932,692.

This application is based on application No. MI95 A 002582 filed inItaly, the content of which is incorporated hereinto by reference.

This invention relates to improved antigenic peptides, methods for thepreparation thereof, and use thereof.

It is known that antibodies recognise proteins important in diagnosticvia binding to specific protein epitopes, formed generally by sequences5 to 20 amino acids long.

In many cases, synthetic peptides corresponding to protein epitopes havebeen used for the preparation of diagnostic kits useful for theidentification and/or the quantitative determination of antibodiesassociated to several pathologies. Identification of protein epitopesrelevant for diagnostic use is carried out by producing, via chemicalsynthesis, peptides corresponding to different portions of the proteinunder examination, and evaluating the antigenic behavior thereof bymeans of a panel of sera of other biological fluids containing thecorresponding antibody.

Very often, protein epitope structure is complex and their synthesisexpensive.

Furthermore, chemical modification in the peptides identified asepitopes may lead, in an unpredictable way, to variations of thecapability of antibody recognition (antigenicity), methadologies ofbroad applicability for improving, by making it more simple, thechemical structure of an antigenic peptide without altering theantigenic properties thereof are not yet available.

Therefore, there is still a great demand of an improvement in thestructure of an antigenic peptide allowing manufacturing it at a lowercost, increasing the stability thereof against proteases, increasing thechemical stability thereof, and/or reducing aspecific interactions withother antibodies (antigenic cross-reactivity), without alteringantigenic properties thereof.

Obviously, these advantages would have repercussions on the productionof diagnostic kits, the use of said antigenic peptides as specificligands for the purification of antibodies for diagnostic or therapyapplications, and on the production of synthetic vaccines.

Surprisingly, it has now been found that these goals are reached whensubstituting one or more amino acids in the antigenic peptide sequenceodd position, or respectively even, position with a glycine residue.

It is therefore a first object of this invention to provide an improvedantigenic peptide, characterized in that the amino acid sequence, bothnormal and retro, is modified by replacing one or more amino acids withglycine, and that, when more than one amino acid is replaced, all thereplaced amino acids are in odd position, or respectively, in evenposition, the amino acids having L or D configuration.

It is therefore a second object of this invention to provide a methodfor improving an antigenic peptide having a known amino sequence,characterized in that at least one amino acid in the sequence isreplaced with glycine and that, when more than one amino acid isreplaced, all the amino acids are replaced in the sequence evenposition, or respectively in the sequence odd position, and in thatimproved antigenic peptide, the amino acids having L or D configuration.

For sake of clarity, an antigenic peptide whose sequence is known andwherein, in the normal or retro sequence, at least one amino acid isreplaced with glycine, will be hereinafter mentioned as “parentantigenic peptide”.

Many are the “parent antigenic peptides” used in diagnostics for thepreparation of kits employed for the determination of specificantibodies in serum and/or other biological fluids. For example, for HIV1 or 2 infection diagnosis, peptides corresponding to the 586-610 envregion of HIV virus are used, while for hepatitis C (HCV) infection indiagnostic kits are used peptides corresponding to regions E1, NS4, andNS5. A list of Chemical Abstract Registry Numbers to antigenic peptides(i.e. “parent antigenic peptides”) which may be improved according tothis invention is shown in the enclosed Table.

It is a third object of this invention to provide a diagnostic kit,characterized in that it contains at least one improved antigenicpeptide as defined above.

It is a fourth object of this invention to provide the production ofmono- or polyclonal antibodies able to recognize a respective “parentantigenic peptide”, characterized in that a suitable animal is immunizedwith a corresponding improved antigenic peptide of this invention, bloodor other suitable tissues are taken after a suitable time and thedesired antibody is isolated according to conventional techniques, suchas chromatography and precipitation. As it is known to the personsskilled in the art, blood is taken for the preparation of polyclonalantibodies, while other tissues, such as the spleen, are taken for thepreparation of polyclonal antibodies.

It is a fifth object of this invention to provide a vaccine,characterized in that it comprises at least one improved antigenicpeptide as defined above.

It is a sixth object of this invention to provide a method for antibodypurification according to the ligands technique, characterized in thatthe ligand is an improved antigenic peptide as defined above.

The selection of the aminoacids to be replaced in odd or in evenposition in the antigenic peptide sequence should be driven by criteriawell known to the persons skilled in the art, such as the cost, thecoupling yield with the preceding and following amino acids, thecomplexity of protection and deprotection of the reactive groups, thechemical and enzymatic stability, and the like.

A typical example of amino acids which may be preferably substitutedwith glycine comprise: cysteine, arginine, asparagine, aspartic acid,glutamic acid, glutamine, histidine, lysine, serine, tyrosine,threonine, and tryptophan.

The improved antigenic peptides according to this invention may beeasily prepared according to conventional techniques of the peptidechemistry which comprise suitable protection of amino acids, coupling ofprotected amino acids and removal of protecting groups.

Preferably, they are prepared according to the solid phase synthesistechnique, which comprises the following steps:

coupling , using suitable reactants, of the first amino acid from theC-terminus protected by suitable groups both in the amino group and,when required, in the side chain, on a suitable support for solid phasesynthesis,

removal of the protecting groups at the amino group with suitablereactants,

coupling of the following amino acid from the C-terminus, protected atthe amino group and, when required, in the side chain,

removal of the protecting group at the amino group and repetition of thecoupling and deprotection cycles until all the amino acids comprised inthe peptide sequence have been assembled,

removal of all remaining side chain protecting groups from the assembledantigenic peptide and cleavage from the resin.

These techniques are widely described in the literature and well knownto the persons skilled in the art. See, for example, Atherton &Sheppard, 1989, Solid Phase Peptide Synthesis: A practical approach, IRLPress, Oxford, UK.

Generally, the diagnostic use of the compounds of this invention inprocedures for antibody determination comprises the formation ofcomplexes with antibodies raised against the “parent antigenic peptide”.

Typically, this technique comprises:

1) immobilizing at least one improved antigenic peptide of thisinvention on plastic ELISA microtiter plates, or on particles useful fordiagnostic applications, either as they are or in form of conjugateswith other molecules;

2) incubating a sample containing the desired antibody with theimmobilized improved antigenic peptide;

3) washing the complex immobilized improved antigenic peptide/antibody;and

4) detecting the adsorbed antibody by means of a suitable reactant.

These techniques are widely described in the literature and are wellknown to the persons skilled in the art. See for example“Immunochemistry in Practice”, Johnstone & Thorpe, (1987) Blackwell,Oxford, UK.

Preferably, step 1 is carried out using plastic microtiter plates, suchas, for example, in PVC, with 96 wells filled with 0.1 M sodiumbicarbonate solutions, pH 9.0, containing variable amounts (from 0 to 50micrograms) of an improved antigenic peptide of this invention,previously conjugated to bovine serum albumin. After 24 hoursincubation, the solution is removed, the microtiter plates are washedwith phosphate buffer (pH 7.3), and the wells are filled with a 3%bovine albumin solution to eliminate sites of aspecific interaction.

In step 2, the microtiter plates are washed with phosphate buffer (pH7.3), and the wells are filled with biological samples containing theantibodies to be determined. The plates are then incubated at 20-37° C.for 4-18 h.

Washing (step 3) is preferably carried out with phosphate buffer (pH7.3).

Finally, step 4 is carried out by adding a solution containing anantibody anti-antibody to be determined (e.g. a rabbit antibody againsthuman immunoglobulins) conjugated to the enzyme peroxidase. After 2 hincubation, the microtiter plates are washed again with phosphate buffer(pH 7.3). A solution of o-phenylenediamine is then added and colorformation is detected using suitable plate readers.

Typically, the preparation of an antibody against an improved antigenicpeptide able to recognize the corresponding “parent antigenic peptide”comprises:

1) immunizing a suitable animal, such as rabbit, sheep, rat, mouse orthe like, with at least a suitable improved antigenic peptide accordingto this invention, possibly conjugated to a carrier proteins such as,for example, albumin and emocianyn, using suitable adjuvants;

2) blood sampling from said animal after a preselected period of timeand separation of the serum fraction;

3) purifying the antibody according to conventional techniques such as,for example, chromatography or precipitation.

Also these techniques are well described in the literature and wellknown to the persons skilled in the art.

Typically the purification of an antibody against an antigenic peptidecomprises the following steps:

1) immobilizing an improved antigenic peptide on a suitable support foraffinity chromatography;

2) packing said support in a suitable chromatographic column;

3) equilibrating said column with a buffer favoring the interactionbetween the immunoglobulin and the antigen;

4) column loading with a fluid containing at least one immunoglobulinable to recognize the “parent antigenic peptide”;

5) column washing with at least one buffer able to elute the impuritieswithout affecting the interaction between immunoglobulin/improvedantigenic peptide; and

6) elution of the previously adsorbed immunoglobulin from the columnusing a suitable dissociating eluent.

Steps 1) to 6) are carried out according to conventional techniques wellknown to the persons skilled in the art.

These and other features of this invention will become more evident fromthe following examples and from the enclosed figures where:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the planar view of the structure of an improved antigenicpeptide of this invention of formula NH₂-Ala-Gly-Leu-Gly-Ala-Gly-COOH(referred to as (−G)P15) (SEQ ID NO:1); corresponding to the parentpeptide P15 where all the amino acids in the even position are replacedwith glycine.

FIG. 2 shows the planar view of the structure of the “parent antigenicpeptide” P15 of formula NH₂-Ala-Asp-Leu-Asp-Ala-Arg-COOH. (SEQ ID NO:2);and

FIG. 3 shows the planar view of the structure of the improved antigenicpeptide of this invention of formula NH₂-Gly-Asp-Gly-Asp-Gly-Arg-COOH(SEQ ID NO:3) (referred to as (+G)P15) corresponding to the “parentantigenic peptide” P15 where all the aminoacids in the odd position arereplaced with glycine.

FIG. 4 is the axial view of the peptide of FIG. 1.

FIG. 5 is the axial view of the peptide of FIG. 2.

FIG. 6 is the axial view of the peptide of FIG. 3.

FIG. 7 shows the interaction between antibodies raised against the“parent antigenic peptide” P15 and peptide P15, improved antigenicpeptides of this invention (−G)P15 and (+G)P15, and an unrelatedpeptide.

FIG. 8 shows that the interaction between antibodies raised against the“parent antigenic peptide” P15 and peptide P15 is inhibited in thepresence of improved antigenic peptides (−G)P14 and (+G)P15 and peptideP15 itself, but not in the presence of unrelated peptides.

FIGS. 1-6 show that the improved antigenic peptide (−G)P1 5 maintainsall the side chains located below the peptide backbone of the “parentantigenic peptide”. The improved antigenic peptide (+G)P15 maintains allthe side chains in the upper part of the parent antigenic P15.

EXAMPLE 1

Preparation of Improved Antigenic Peptides and Determination of TheirAntigenic Properties

Polyclonal antibodies have been prepared by rabbit immunization with the“parent antigenic peptide” P15 (NH₂-Ala-Asp-Leu-Asp-Ala-Arg-COOH)conjugated to bovine serum albumin.

The corresponding improved antigenic peptides have also been prepared.

A first improved antigenic peptide has been obtained replacing theresidues in even position with glycine. The corresponding peptide havingthe sequence NH₂-Ala-Gly-Leu-Gly-Ala-Gly-COOH has thus been obtained,which is hereinafter referred to as (−G)P15.

A second improved antigenic peptide has been obtained via replacement ofthe residues in the odd position with glycine. The corresponding peptidehaving sequence NH₂-Gly-Asp-Gly-Asp-Gly-Arg-COOH has been thus obtained,which is hereinafter referred to as (+G)P15.

Compounds P15, (+G)P15, and (−G)P15 have been synthesized with anautomatic peptide synthesizer (ABI 431-A, software version 1.1,Perkin-Elmer, U.S.A.) following the protocols suggested by themanufacturer and based on methodologies described in the literature(Atherton & Sheppard, 1989, Solid Phase Peptide Synthesis: A practicalapproach, IRL Press, Oxford, UK).

The synthesis has been carried out starting from a acid-labile resin forpeptide synthesis prederivatized with glycine (Novabiochem Cat. No.04-12-2800, 0.1 mmole) for (−G)P15 peptide and from arginine(Novabiochem Cat. No. 04-12-2850, 0.1 mmol) for antigenic peptides P15and (+G)P15. Both glycine and arginine were protected at the N-terminalamino group a Fmoc group which, in the first synthesis cycle, has beenremoved by treatment with 3.0 ml of piperidine (20% inN-methyl-pyrrolidone)(ABI Cat No. 400629) for 14 minutes at roomtemperature under stirring.

The deprotected resin has been then washed 5 times with 2.5 ml ofN-methyl-2-pyrrolidone (Merck Cat. No. 806072) for 9 minutes undershaking at room temperature.

In the meantime the amino acid residue in position 2 from the C-terminus(1 mmole) has been preactivated by incubation with 1 ml of HOBt 1 Mdissolved in N-methyl-2-pyrrolidone (ABI Cat. No. 400662) and 1 ml of 1M dicyclohexylcarbodiimide in N-methyl-2-pyrrolidone (ABI Cat. No.400663).

The activated amino acid has been then incubated with the resin for 51minutes under constant shaking. After washing withN-methyl-2-pyrrolidone (4 washes for 0.5 minutes with 2 ml) the resinunderwent a further deprotection cycle with piperidine and a furthercoupling cycle with the next amino acid. This sequential steps procedurehas been repeated until all the amino acid residues have been assembled.In detail, the following amino acid derivatives have been used:Fmoc-Asp(tBu) (Novabiochem Cat. No. 04-12-1013), Fmoc-Ala (NovabiochemCat. No. 04-12-1006); Fmoc-Leu (Novabiochem Cat. No. 04-12-1025. Aftercompletion of synthesis cycles, and removal of the N-terminal Fmoc groupby piperidine treatment, the resin has been washed with methanol,dichloromethane, and again with methanol and carefully dried undervacuum for 12 h. Afterwards, the peptide has been detached from theresin by incubation of 100 mg of resin with 5 ml of a mixture oftrifluoroacetic acid (Pierce Cat. No.28901)/phenol (Carlo Erba Cat. No.451285)/water (Merck Cat. No.15333) ethanedithiol (Aldrich Cat No. E360-0)/thioanisol (Aldrich Cat. No. T 2,800-2) 84:4:3:3:3 v/v for 2 h atroom temperature under shaking. The resin has been then filtered using asintered glass filter. The filtrate has been reduced in volume to few mlby vacuum evaporation and the residual liquid has been treated with 50ml of cold ethyl ether. The precipitated peptidic material has been thenseparated by centrifugation and the centrifuged material resuspended in25 ml of water/acetonitrile/TFA 50:50:0.1, frozen and lyophilized. Atthe end, the lyophilized material has been purified from contaminants byhigh performance liquid chromatography (HPLC) using a Lichrospher RP-8column (25×1 cm I.D.) (Merck Cat. No. 150153), equilibrated at a flowrate of 3 ml/min with water/acetonitrile/TFA 95/5/0.1, and eluting witha linear gradient of acetonitrile ranging from 5% to 80% in 55 minutes.Material corresponding to the main peak has been collected, frozen, andlyophilized.

The antigenic peptides thus obtained have been conjugated to bovineserum albumin using glutaraldheyde according to known procedures(Immunochemistry in Practice, Johnstone & Thorpe, (1987) Blackwell,Oxford, UK), dialyzed against 0.1 M acetic acid, and lyophilized.

Microtiter plates for ELISA determination (Falcon Cat. No. 3912) havebeen treated with the peptides conjugated to BSA in concentration offrom 0 to 50 μg/ml in a sodium bicarbonate buffer pH 9.0, for 12 h at 4°C. The plates have been washed with a 150 mM sodium phosphate buffer, pH7.2 (PBS) and then, to each well of the plate, 200 microliters of a PBSsolution containing 3% bovine serum albumin (BSA, Sigma Cat. NO. A-4503)have been added.

After one hour, the plates have been washed again 4 times with PBS, andeach well has been filled with solutions (100 μl) containing rabbitantibodies against peptide P15, in concentration of from 0 to 20 μg/ml.After 2 hours incubation, plates have been washed 8 times with PBS andthen in each well 100 μl of a solution containing anti-rabbitimmunoglobulin goat immunoglobulins conjugated to peroxidase (Sigma Cat.No. A-6154) diluted 1:100 with PBS containing 3% BSA have been added.After one hour, the plates have been washed again with PBS. Then eachwell has been filled with 100 μl of a solution containing 1 mg/ml ofo-phenylenediamine (Sigma P 6912) in 0.1 M sodium citrate, and 5 mMhydrogen peroxide. After 30 minutes, the reaction has been stopped byadding 25 microliters of 4.5 M sulfuric acid. The absorbance at 490 nmof each well has been determined using a plate reader (Merck, Mod.MIOS). The results are shown in FIG. 7.

The specificity of the interactions observed has been determined byinhibition studies, via incubation of the antibody directed againstpeptide P15 with peptides P15, (−G)P15, (+G)P15 at varyingconcentrations.

As shown in FIG. 7, immunoglobulins became associated to the antigenicpeptides P15, (−G)P15, and (+G)P15, immobilized on the plate, in a dosedependent fashion and with about the same intensity. The interaction isinhibited in the presence of peptides P15, (−G)P15, and (+G)P15 insolution in a dose dependent manner and specifically while controlpeptides (BSA, or an unrelated peptide) do not inhibit the interaction(FIG. 8).

Similar recognition properties have been shown by improved antigenicpeptides of this invention (−G)P15 and (+G)P15 when produced from aminoacids having all D configuration, and by improved antigenic peptides ofthis invention having reversed sequence direction. Indeed, peptidesNH₂-Gly-Ala-Gly-Leu-Gly-Ala-COOH, corresponding to peptide (−G)P15 withreversed sequence, and NH₂-Arg-Gly-Asp-Gly-Asp-Gly-COOH, correspondingto peptide (+G)P15 with reversed sequence, both in configuration all Lor all D, showed similar functional properties.

EXAMPLE 2

Purification of Anti-P15 Antibodies on Columns Prepared By Immobilizing(−G)P15 and (+G)P15

The improved antigenic peptides (−G)P15 and (+G)P15 (5 mg) of thisinvention have been dissolved separately in 5 ml of a 0.1 M sodiumbicarbonate buffer, pH 9.0, and then added to 1.2 g of Eupergit C30 Nresin (Sigma Cat. No. 0 9879), affinity chromatography supportpreactivated with epoxyde groups for the direct coupling of peptides andproteins, respectively. The suspension has been shaken for 24 h and thecoupling extent has been monitored by sampling of the reaction mixturefollowed by RP-HPLC analysis. In both cases, about 85% of initialpeptide was covalently coupled to the support after 24 h.

The derivatized resins have then been washed with 50 ml of 1 M Tris pH9.0, and packed on glass columns 100×6.6 mm I.D. For purificationprocedures, the columns have been equilibrated with Tris buffer 50 mM,pH 7.0, at a flow rate of 1 ml/min. while monitoring the effluent at 280nm. On the two columns, 1 ml of crude rabbit serum, containingantibodies against peptide P15, has been applied and, after elution ofnon retained material at the column void volume, the eluent has beenchanged to 0.1 M acetic acid.

Material desorbed by such treatment has been collected and analyzed byelectrophoretic analysis on polyacrylamide gels. In both cases, thecolumns have been able to purify efficiently the anti-P15 antibodies inthe serum. Similar recognition properties have been showed by theimproved antigenic peptides of this invention (−G)P15 and (+G)P15produced from amino acids having D configuration, as well as by improvedantigenic peptides of this invention having reversed sequence. Indeed,both peptides NH₂-Gly-Ala-Gly-Leu-Gly-Ala-COOH, corresponding to peptide(−G)P15 with reversed sequence, and NH₂-Arg-Gly-Asp-Gly-Asp-Gly-COOH,corresponding to peptide (+G)P15 with reversed sequence, showed similarfunctional properties, both in configuration all L or all D.

EXAMPLE 3

Production of Antibodies Against (−G)P15 and (+G)P15 in Rabbits

Improved antigenic peptides of this invention (−G)P15 and (+G)P15conjugated to BSA, prepared as previously described, have been used forrabbits (New Zealand) immunization by injections in the rear foot pads.After 4 weeks, a subsequent boost with the same improved antigenicpeptides has been carried on, and after 2 months blood samples have beentaken for the preparation of the serum fraction.

As detected using ELISA technique as described above and usingmicrotiter plates for ELISA determinations sensitized with peptide P15,sera obtained from the two different immunizations were able torecognize peptide P15 showing an affinity and selectivity degreecomparable to those shown by the antiserum against parent peptide P15.

Similar recognition properties have been shown by improved antigenicpeptides of this invention (−G)P15 and (+G)P15 produced from amino acidshaving D configuration, as well as by improved antigenic peptides ofthis invention having reversed sequence.

Indeed, both peptides NH₂-Gly-Ala-Gly-Leu-Gly-Ala-COOH, corresponding topeptide (−G)P15 with reversed sequence, andNH₂-Arg-Gly-Asp-Gly-Asp-Gly-COOH, corresponding to peptide (+G)P15 withreversed sequence, both in configuration all L or all D, showed similarfunctional properties.

TABLE 121478-87-3 131143-43-6 119330-55-1 119330-56-2 119330-57-3131143-38-9 134908-81-9 134908-82-0 134908-79-5 134908-80-8 129403-71-0133334-08-4 132965-90-3 115283-36-8 117628-43-0 118389-56-3 119699-13-7119699-12-6 119699-14-8 119699-15-9 119699-16-0 119699-17-1 126729-43-9131200-77-6 131093-03-3 131280-39-2 126546-16-5 126546-17-6 126546-19-8126546-14-3 126546-15-4 126546-18-7 134547-78-7 121272-75-1 121272-70-6128513-74-6 128513-76-8 128513-75-7 128513-52-0 128513-80-4 128513-77-9128513-78-0 127496-43-9 128027-09-8 127496-49-5 127496-48-4 127496-47-3124148-04-5 128027-04-3 127496-50-8 129710-61-8 129710-60-7 129710-63-0134945-29-2 132866-87-6 130037-19-3 130037-20-6 103351-42-4 134145-91-8118113-84-1 119440-22-1 114680-04-5 123608-06-0 123608-07-1 121683-16-7121683-17-8 123608-08-2 123608-09-3 124024-82-4 124024-83-5 124024-84-6124024-85-7 94361-68-9 86025-40-3 107720-45-6 107720-46-7 117276-07-086025-41-4 117536-69-3 87608-92-2 99731-72-3 121683-18-9 121683-19-0123608-10-6 114355-74-7 114355-75-8 114355-76-9 114355-77-0 114355-78-1114355-79-2 114355-80-5 114355-81-6 114355-82-7 112195-83-2 118103-21-2118103-22-3 118103-23-4 118103-24-5 106440-45-3 106440-43-1 106440-44-2123056-69-9 123608-11-7 123608-12-8 137544-82-2 129773-36-0 129403-69-6129403-68-5 129403-77-6 134145-93-0 134145-94-1 134145-92-9 134145-95-2134145-96-3 134145-97-4 134145-98-5 134146-64-8 134145-99-6 134146-00-2134146-01-3 134146-02-4 134146-63-7 134146-62-6 134146-65-9 129403-76-5132654-55-8 121256-87-9 136627-97-9 133134-51-7 133134-50-6 121478-86-2108421-86-9 119330-53-9 119330-54-0 119330-52-8 136799-67-2 123938-62-5110157-83-0 126729-44-0 121546-71-2 133134-53-9 133134-54-0 133134-52-8133134-56-2 133134-55-1 118113-88-5 118113-89-6 124542-66-1 133134-57-3137085-93-9 137085-94-0 136361-96-1 130730-37-9 130730-38-0 130730-35-7130730-36-8 131333-19-2 116783-38-1 126729-45-1 132989-34-5 132989-35-6134547-80-1 134547-81-2 130527-74-1 134773-23-2 134146-03-5 134773-24-3133107-89-8 122096-66-6 122096-67-7 127712-26-9 105080-81-7 134773-25-4134773-26-5 126902-84-9 126902-85-0 128794-23-0 126467-93-4 108778-09-2118901-31-8 123626-27-7 118901-30-7 125006-38-4 128511-32-0 128511-28-4128511-29-5 128511-31-9 128511-30-8 130809-05-1 130809-06-2 130809-07-3130809-08-4 129804-69-9 129804-68-8 129804-71-3 129804-70-2 130809-09-5130809-10-8 130809-11-9 125807-38-7 125807-37-6 134773-28-7 128393-14-6128393-15-7 136251-99-5 115250-87-8 115250-85-6 126039-23-4 134012-76-3130843-53-7 125052-49-5 125052-51-9 125913-27-1 125052-54-2 125052-55-3125052-56-4 125052-57-5 125052-58-6 125913-25-9 125912-55-2 125913-05-5125913-26-0 125934-29-4 125934-75-0 125913-28-2 128122-97-4 125912-48-3125912-61-0 125934-21-6 125912-57-4 125934-25-0 125913-24-8 118842-09-4118842-10-7 121272-71-7 124670-01-5 136894-52-5 128907-18-6 128907-19-7128907-20-0 128907-21-1 129069-07-4 129069-08-5 129069-09-6 128393-16-8126467-94-5 126467-95-6 131198-28-2 110908-53-7 110908-54-8 126467-96-7126467-97-8 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162032-95-3 162032-96-4 162032-98-6162032-93-1 162032-97-5 162032-99-7 162629-36-9 162033-00-3 162033-01-4162455-90-5 162455-98-3 162456-04-4 162455-03-6 162455-89-2 162455-97-2162456-03-3 162455-74-5 162455-85-8 162455-92-7 162456-00-0 162456-06-6162455-84-7 162455-91-6 162455-99-4 162456-05-5 162455-76-7 162458-02-2162455-75-6 162456-01-1 162456-08-8 162456-07-7 162456-10-2 162456-09-9162455-86-9 162455-94-9 162455-93-8 162455-88-1 162455-96-1 162455-77-8162455-87-0 162455-95-0 162455-78-9 162455-80-3 162455-79-0 162455-81-4162455-82-5 149224-09-9 162456-12-4 162456-11-3 162456-14-6 162456-13-5161414-83-1 161414-84-2 161414-85-3 160227-93-0 161415-00-5 162875-32-3163480-83-9 159575-21-0 160870-79-1 160339-90-2 160339-92-4 160339-91-3162077-61-4 163482-34-6 160339-93-5 161850-11-9 162077-64-7 160870-88-2161704-96-7 160871-27-2 162876-18-8 161026-74-0 161347-80-4 161347-81-5161347-75-7 161347-72-4 161347-73-5 161347-78-0 161347-77-9 161347-79-1161347-71-3 161347-76-8 161347-74-6 162535-81-1 102484-07-1 162063-98-1161704-90-1 160870-87-1 161704-91-2 162063-90-3 144132-38-7 160829-87-8159575-22-1 160870-83-7 160870-84-8 159994-33-9 160870-77-9 160870-78-0160966-18-7 131143-43-6 138988-32-6 160871-07-8 159669-58-6 157298-43-6157858-32-7 162164-29-6 157858-33-8 157858-34-9 163479-70-7 157858-31-6162164-30-9 162952-75-2 162952-76-3 160872-31-1 161172-88-9 161704-61-6159075-77-1 159075-79-3 142931-01-9 159075-76-0 159075-78-2 160871-20-5160871-18-1 160871-19-2 160871-21-6 160871-22-7 160871-23-8 162568-70-9162568-71-0 161706-45-2 156289-38-2 160227-94-1 160227-95-2 161347-85-9161347-84-8 161347-83-7 162063-91-4 162063-92-5 162063-93-6 162063-94-7162063-95-8 162063-96-9 162456-38-4 160026-03-9 160026-02-8 163479-88-7161706-52-1 163479-64-9 160403-10-1 161706-51-0 161738-85-8 122191-00-8160871-06-7 161414-03-5 199965-54-5 161310-02-7 162033-12-7 162064-19-9161347-42-8 161348-09-0 161347-56-4 151880-78-3 162079-90-5 162079-89-2159318-44-2 159204-57-6 152572-66-9 160275-60-5 160275-61-6 160830-98-8120177-71-1 146047-11-2 160276-29-9 160276-30-2 159522-88-0 157091-78-6157091-86-6 157091-93-5 157091-87-7 157091-88-8 157091-89-9 157091-90-2157091-91-3 160478-27-3 161026-94-4 159522-87-9 159607-90-6 159577-11-4162996-08-9 138157-22-9 156201-48-8

3 6 amino acids amino acid single linear peptide unknown 1 Ala Gly LeuGly Ala Gly 1 5 6 amino acids amino acid single linear peptide unknown 2Ala Asp Leu Asp Ala Arg 1 5 6 amino acids amino acid single linearpeptide unknown 3 Gly Asp Gly Asp Gly Arg 1 5

What is claimed is:
 1. A method of modifying an antigenic peptide havinga known amino acid sequence, comprising replacing more than one, but notall, the amino acids in odd positions, or respectively, in evenpositions, of the normal or retro sequence of said known amino acidsequence of the antigenic peptide with glycine, and the amino acid otherthan glycine have L or D configuration.